JP4379318B2 - Optical measuring apparatus and optical measuring method - Google Patents
Optical measuring apparatus and optical measuring method Download PDFInfo
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Description
本発明は、液体試料を光学的に測定する光学的測定装置および方法に関し、さらに詳細には、液体中に２種類以上の粒子が混在する液体試料について測定する光学的測定装置および方法に関する。本発明の光学的測定装置および方法は、例えば、異なる粒径の蛋白質分子等が混在する溶液中の粒子存在比の確認、さらには粒子濃度、粒形、粒径の測定に適用することができる。 The present invention relates to an optical measurement apparatus and method for optically measuring a liquid sample, and more particularly to an optical measurement apparatus and method for measuring a liquid sample in which two or more kinds of particles are mixed in the liquid. The optical measuring apparatus and method of the present invention can be applied to, for example, confirmation of the particle abundance ratio in a solution containing protein molecules having different particle diameters, and further measurement of particle concentration, particle shape, and particle diameter. .
近年、液体中で粒子を計測することは、例えば蛋白質などの生体高分子に関する情報を計測する手段のひとつとして注目されている。
生体高分子を粒子としてみたときの生体高分子の移動しやすさ、すなわち拡散しやすさは、生体高分子の大きさ、形状、結合状態等に依存して変化するので、拡散しやすさを評価することにより、生体高分子に関する種々の情報、例えば粒径や、粒形、結合状態等を知ることができる。
In recent years, measuring particles in a liquid has attracted attention as one of means for measuring information about biopolymers such as proteins.
When the biopolymer is viewed as particles, the ease of movement of the biopolymer, that is, the ease of diffusion, changes depending on the size, shape, binding state, etc. of the biopolymer. By evaluating, it is possible to know various information related to the biopolymer, such as particle size, particle shape, bonding state, and the like.
液体中の粒子の拡散しやすさを計測する手法としては例えば顕微蛍光相関分光法がある（特許文献１参照）。
顕微蛍光分光法によれば、計測対象となる粒子（生体高分子）を蛍光分子で標識処理し、顕微鏡視野下でこれを励起照明し、蛍光を発する計測対象粒子のブラウン運動に伴う蛍光強度変化を計測（蛍光粒子の数をカウントする）して、計測対象粒子の拡散係数を求めるものである。
As a method for measuring the ease of diffusion of particles in a liquid, for example, there is a microscopic fluorescence correlation spectroscopy (see Patent Document 1).
According to microscopic fluorescence spectroscopy, the measurement target particles (biopolymers) are labeled with fluorescent molecules, and this is excited and illuminated in the microscope field. The fluorescence intensity changes due to the Brownian motion of the measurement target particles that emit fluorescence. Is measured (the number of fluorescent particles is counted) to obtain the diffusion coefficient of the measurement target particles.
また、出願人自身による先行特許出願において、標識化を行うことなく粒子（溶解したもの又は分散したもの）の拡散しやすさを計測するための光学的測定装置として、液体中の粒子に誘電泳動を生じさせて移動することにより、粒子集中領域を形成し、その後、誘電泳動を停止して粒子集中領域から粒子を拡散させたときの屈折率変化から、粒子の拡散に関する評価を行う装置を提案している（特願２００４−２０４０２４号）。この光学的測定装置では、２本の平行に並ぶ電極を通じて被測定溶液に電圧を印加して誘電泳動を引き起こすことにより、溶液の局所的な屈折率変化を発生させている。 In addition, in an earlier patent application filed by the applicant himself, as an optical measuring device for measuring the ease of diffusion of particles (dissolved or dispersed) without labeling, dielectrophoresis is performed on the particles in the liquid. Produces a device that evaluates the diffusion of particles from the change in refractive index when the particle concentration region is formed by moving the particles and then the dielectrophoresis is stopped and the particles are diffused from the particle concentration region (Japanese Patent Application No. 2004-204024). In this optical measuring apparatus, a local refractive index change of the solution is generated by applying a voltage to the solution to be measured through two parallel electrodes to cause dielectrophoresis.
また、出願人による他の先行特許出願において、基本回折光パターンを生じさせる回折格子に、所定の交流電圧を印加して粒子に誘電泳動を起こさせることにより、基本回折光パターンとは異なる変形回折光パターンを発生させ、変形回折光パターンに基づいて液体中の粒子に関する情報を計測することを提案している（特願２００４−２４１９０７号）。
上述した特許文献１に開示された顕微蛍光相関分光法による光学的測定では、試料を標識する必要があり、そのための煩わしい前処理作業を行わなければならない。
また、顕微蛍光相関分光法により測定すれば、粒子に標識化処理を施してしまうので、粒子を完全な自然状態で測定することはできない。
これに対し、上述した誘電泳動による変形回折光パターンに基づいて液体中の粒子の光学的測定を行う方法では、標識化処理の必要がないので、前処理の煩わしさがなく、また、粒子を完全な自然状態で測定することができる。
In the optical measurement by the micro-fluorescence correlation spectroscopy disclosed in Patent Document 1 described above, it is necessary to label the sample, and it is necessary to perform troublesome pre-processing work.
Further, if the measurement is performed by micro-fluorescence correlation spectroscopy, the particles are labeled, so that the particles cannot be measured in a completely natural state.
On the other hand, in the method of optically measuring particles in a liquid based on the above-described deformed diffraction light pattern by dielectrophoresis, there is no need for labeling, so there is no troublesome pretreatment, It can be measured in a completely natural state.
しかしながら、蛋白質分子を粒子とする試料の場合をはじめ、液体中に含まれる粒子が２種類、あるいはそれ以上である場合がある。また、蛋白質分子が１種類であっても、結合状態が変化することにより蛋白質分子の形状が異なり、実質的に異なる２種類の粒子となる場合もある。
実質的に異なる２種類以上の粒子が含まれる試料の場合に、単に交流電圧を印加して誘電泳動を発生させ、安定状態になったときの変形回折光パターンを測定する方法では、２種類以上の粒子が混在した状態での情報を得ることができるが、それぞれの種類の粒子の濃度、特性を分離して測定することができなかった。
However, there may be two or more kinds of particles contained in the liquid, including a sample containing protein molecules as particles. Moreover, even if there is only one type of protein molecule, the shape of the protein molecule is different due to a change in the binding state, which may result in two substantially different types of particles.
In the case of a sample containing two or more types of particles that are substantially different, two or more types are used in the method of measuring the deformed diffracted light pattern when a stable state is obtained by simply applying an alternating voltage to generate dielectrophoresis. Although it is possible to obtain information in a state where the particles are mixed, it is impossible to measure the concentration and characteristics of each type of particles separately.
そこで、本発明は標識化のための前処理を行うことなく、試料液体中の粒子の状態を計測する光学的測定装置および方法を提供することを目的とする。
さらに、試料液体中に２種類あるいはそれ以上の数の異なる種類の粒子が存在した場合に、異なる粒子ごとの情報を分離して得ることができる光学的測定装置および方法を提供することを目的とする。
Therefore, an object of the present invention is to provide an optical measurement apparatus and method for measuring the state of particles in a sample liquid without performing pretreatment for labeling.
It is another object of the present invention to provide an optical measuring apparatus and method capable of separately obtaining information for different particles when two or more different types of particles are present in a sample liquid. To do.
上記課題を解決するためになされた本発明の光学的測定装置は、光源と、印加電圧値を変化することができる交流電源と、液体試料を保持する容器と、容器内の液体試料と接する位置に形成され、光源から光が照射されることにより基本回折光パターンを生じる回折格子と、回折格子の少なくとも一部を構成するとともに、交流電源から交流電圧を印加することが可能な電極対と、電極対に印加する電圧値を変化させる電圧制御部と、回折格子による回折光を検出する光検出器とを備え、電極対に電圧を印加して液体試料の屈折率分布を変化することにより、基本回折光パターンとは異なる変形回折光パターンを発生させるとともに、電圧制御部により印加電圧値が次第に大きくなるように変化させたときの変形回折光パターン強度の変化に基づいて液体試料に関する情報を計測するようにしている。 The optical measuring device of the present invention made to solve the above problems is a light source, an AC power source capable of changing an applied voltage value, a container holding a liquid sample, and a position in contact with the liquid sample in the container. A diffraction grating that generates a basic diffracted light pattern by being irradiated with light from a light source, and an electrode pair that constitutes at least a part of the diffraction grating and is capable of applying an AC voltage from an AC power source, A voltage control unit that changes the voltage value applied to the electrode pair and a photodetector that detects diffracted light by the diffraction grating, and by applying a voltage to the electrode pair to change the refractive index distribution of the liquid sample, A modified diffracted light pattern that is different from the basic diffracted light pattern is generated, and the intensity of the deformed diffracted light pattern changes when the applied voltage value is gradually increased by the voltage controller. And so as to measure the information about the liquid sample Zui.
この発明によれば、容器内に液体試料、特に、２種類以上の異なる粒子を含んだ液体試料を入れて保持した状態で、回折構成を構成する電極対に向けて光源から光を照射する。このとき、回折格子により光が回折され、回折光パターンを生じる。このときの回折光パターンが基本回折光パターンとなる。
そして、電源から電極対に交流電圧を印加して誘電泳動を発生する。誘電泳動により粒子が移動すると回折格子近傍の液体試料の屈折率が変化し、基本回折光パターンを生じる回折格子とは異なる派生的な回折格子が発生し、変形回折光パターンが発生する。この変形回折光パターンを光検出器で測定することにより、液体試料中の粒子による影響を変形回折光パターンの強度変化として測定することができる。
このときの電極対への印加電圧値を電圧制御部により制御する。この印加電圧制御について説明する。
誘電泳動中に微粒子に働く力はブラウン運動による等方的な拡散力と誘電泳動力の和と考えることができるが、電極にかかる電圧値に関わらず、ブラウン運動による拡散力は一定である。一方、粒子を引き寄せる誘電泳動力に注目すると、その力は分極による双極子モーメントと電界勾配に比例する。つまり誘電泳動力は印加電圧の２乗に比例することになる。
According to this invention, light is emitted from the light source toward the electrode pair constituting the diffractive structure in a state where a liquid sample, particularly a liquid sample containing two or more kinds of different particles is placed and held in the container. At this time, light is diffracted by the diffraction grating to generate a diffracted light pattern. The diffracted light pattern at this time becomes the basic diffracted light pattern.
Then, an AC voltage is applied from the power source to the electrode pair to generate dielectrophoresis. When the particles move by dielectrophoresis, the refractive index of the liquid sample near the diffraction grating changes, and a derivative diffraction grating different from the diffraction grating that generates the basic diffraction light pattern is generated, and a modified diffraction light pattern is generated. By measuring this deformed diffracted light pattern with a photodetector, the influence of particles in the liquid sample can be measured as a change in the intensity of the deformed diffracted light pattern.
The voltage applied to the electrode pair at this time is controlled by the voltage controller. This applied voltage control will be described.
The force acting on the fine particles during the dielectrophoresis can be considered as the sum of the isotropic diffusion force due to the Brownian motion and the dielectrophoretic force, but the diffusion force due to the Brownian motion is constant regardless of the voltage value applied to the electrode. On the other hand, paying attention to the dielectrophoretic force attracting particles, the force is proportional to the dipole moment and electric field gradient due to polarization. That is, the dielectrophoretic force is proportional to the square of the applied voltage.
誘電泳動を生じさせるための交流電圧を印加した場合に、電極に印加する電圧値が小さすぎると誘電泳動力に起因する粒子捕集力が小さいことから、実質的に粒子が集中できない印加電圧値範囲が存在する。すなわち拡散力が誘電泳動力より強くなる印加電圧値範囲があり、この印加電圧値範囲は物質によって異なり、２種類以上の粒子が存在すると、それぞれの粒子ごとに、拡散力が誘電泳動力より強くなる印加電圧値範囲が異なる。 When an alternating voltage for causing dielectrophoresis is applied, if the voltage applied to the electrode is too small, the particle trapping force due to the dielectrophoretic force is small, so that the applied voltage value at which particles cannot concentrate substantially A range exists. That is, there is an applied voltage value range in which the diffusive force is stronger than the dielectrophoretic force, and this applied voltage value range varies depending on the substance. When two or more types of particles exist, the diffusive force is stronger than the dielectrophoretic force for each particle. Different applied voltage value ranges.
この拡散力が誘電泳動力より強くなる印加電圧値範囲が異なることを利用して、粒子を分離する。すなわち、印加電圧値を０から（後述する第１臨界点の予測がつく場合はそれよりも十分に小さい値からでよい）次第に大きくしていく。やがて、誘電泳動力と拡散力との差が最も小さいいずれかの粒子(第１の粒子)の誘電泳動力が拡散力より強くなる境界値（第１臨界点）を超えると、その粒子が最初に捕集されるようになる。このとき、その他の種類の粒子については、まだ誘電泳動力より拡散力が勝っているので捕集されない。
さらに印加電圧値を大きくしていき、誘電泳動力と拡散力との差が次に小さい第２の粒子についての誘電泳動力が拡散力より強くなり、第２の境界値（第２臨界点）を超えると、第２の粒子についても捕集されるようになる。同様に、粒子の種類が３つ以上あるときは、印加電圧値を引き続き大きくして、第３臨界点以降も越えるようにすると、順次、誘電泳動力と拡散力との差が小さい粒子から捕集されるようになる。
The particles are separated using the fact that the applied voltage value range in which the diffusion force is stronger than the dielectrophoretic force is different. That is, the applied voltage value is gradually increased from 0 (if a first critical point described later can be predicted, it may be a value sufficiently smaller than that). Eventually, when the dielectrophoretic force of one of the particles (first particle) having the smallest difference between the dielectrophoretic force and the diffusing force exceeds the boundary value (first critical point) at which the dielectrophoretic force becomes stronger than the diffusing force, the particle first Will be collected. At this time, other types of particles are not collected because the diffusion force is still superior to the dielectrophoretic force.
The applied voltage value is further increased, and the dielectrophoretic force for the second particle having the next smallest difference between the dielectrophoretic force and the diffusing force becomes stronger than the diffusing force, and the second boundary value (second critical point). If it exceeds, the second particles are also collected. Similarly, when there are three or more types of particles, the applied voltage value is continuously increased to exceed the third critical point, and the difference between the dielectrophoretic force and the diffusive force is sequentially captured from the particles. It will be gathered.
このように２種類以上の粒子が存在する場合に、印加電圧値を次第に大きくしていくことにより、捕集される粒子の種類を１つずつ追加することができる。
そこで、印加電圧値を変化させながら、光検出器により、変形回折光パターンの強度変化（回折光強度変化）を測定すると、第１臨界点から第２臨界点までは第１の粒子のみによる変形回折光パターン、第２臨界点から第３臨界点までは、第１の粒子と第２の粒子とによる変形回折光パターンが形成される。２つの粒子による変形回折光パターンの場合の回折光強度は、それぞれの粒子による変形回折光パターンの回折光強度の和であるので、第１臨界点から第２臨界点までの変形回折光パターンによるデータと、第２臨界点から第３臨界点までの変形回折光パターンによるデータとから、第１の粒子による変形回折光パターンの回折光強度と第２の粒子による変形回折光パターンの回折光強度とを分離することができる。さらに、粒子の種類数が増加しても同様の手順で分離することができる。このようにして粒子の種類ごとに分離された変形回折光パターンの回折光強度から粒子ごとの情報を得るようにする。
As described above, when two or more kinds of particles are present, the kind of particles to be collected can be added one by one by gradually increasing the applied voltage value.
Therefore, when the intensity change (diffracted light intensity change) of the deformed diffracted light pattern is measured by the photodetector while changing the applied voltage value, the deformation from only the first particle from the first critical point to the second critical point is performed. From the second critical point to the third critical point, a diffracted light pattern is formed by the first particles and the second particles. Since the diffracted light intensity in the case of the deformed diffracted light pattern by two particles is the sum of the diffracted light intensities of the deformed diffracted light patterns by the respective particles, it depends on the deformed diffracted light pattern from the first critical point to the second critical point. From the data and the data of the modified diffracted light pattern from the second critical point to the third critical point, the diffracted light intensity of the deformed diffracted light pattern by the first particles and the diffracted light intensity of the deformed diffracted light pattern by the second particles And can be separated. Furthermore, even if the number of types of particles increases, it can be separated by the same procedure. Thus, information for each particle is obtained from the diffracted light intensity of the deformed diffracted light pattern separated for each type of particle.
本発明によれば、２種類以上の粒子が含まれる液体試料の光学的測定において、標識化等の煩雑な前処理を行うことなく、試料液体中の粒子の状態を計測することができる。
さらに、試料液体中の２種類あるいはそれ以上の数の異なる種類の粒子による変形回折光パターン強度（変形回折光パターンの回折光強度）を、それぞれ粒子ごとに分離して計測することができるので、混在した粒子の情報だけではなく、粒子ごとの情報を得ることができる。
According to the present invention, in the optical measurement of a liquid sample containing two or more kinds of particles, the state of the particles in the sample liquid can be measured without performing complicated pretreatment such as labeling.
Furthermore, the deformation diffracted light pattern intensity (diffracted light intensity of the deformed diffracted light pattern) by two or more different kinds of particles in the sample liquid can be measured separately for each particle, Not only the information on the mixed particles but also the information for each particle can be obtained.
（その他の課題を解決するための手段および効果）
上記発明において、電極対は、交流電圧が印加されたときに正の電極と負の電極とが隣接する部分が、回折格子周期の２倍以上の整数倍の周期で繰り返すように配置されるようにしてもよい。
この発明によれば、変形回折光パターンにおいて、新たに追加された回折光は、基本回折光パターンでは回折光が存在していなかった中間付近の暗い位置（暗部分）に発生するので、明暗のコントラストがはっきりする位置でピークを検出することができる。
(Means and effects for solving other problems)
In the above invention, the electrode pair is arranged so that a portion where the positive electrode and the negative electrode are adjacent to each other when an AC voltage is applied is repeated at a cycle that is an integral multiple of twice or more the diffraction grating cycle. It may be.
According to the present invention, the newly added diffracted light in the modified diffracted light pattern is generated at a dark position (dark part) near the middle where no diffracted light was present in the basic diffracted light pattern. A peak can be detected at a position where the contrast is clear.
また、上記課題を解決するために別の観点からなされた本発明の光学的測定方法は、光源と、印加電圧値を変化することができる交流電源と、液体試料を保持する容器と、容器内の液体試料と接する位置に形成され、光源から光が照射されることにより基本回折光パターンを生じる回折格子と、回折格子の少なくとも一部を構成するとともに、交流電源から正負の電圧を印加することが可能な電極対と、回折格子による回折光を検出する光検出器とを備え、電極対に交流電圧を印加して誘電泳動により液体試料の屈折率分布を変化させて基本回折光パターンとは異なる変形回折光パターンを発生させ、変形回折光パターンから液体試料中の粒子に関する情報を計測する光学的測定方法であって、少なくとも２種類の異なる粒子が混在する液体試料を容器内に入れ、印加電圧値を次第に大きい電圧値になるように連続的に変化させながら電極対に印加して、そのときの変形回折光パターン強度の変化のデータに基づいて液体中に含まれる粒子に関する情報を計測するようにする。 Further, the optical measurement method of the present invention made from another point of view to solve the above problems includes a light source, an AC power source capable of changing an applied voltage value, a container holding a liquid sample, A diffraction grating that is formed at a position in contact with the liquid sample and generates a basic diffraction light pattern when irradiated with light from a light source, and at least part of the diffraction grating, and a positive and negative voltage is applied from an AC power source A basic diffracted light pattern by applying an alternating voltage to the electrode pair and changing the refractive index distribution of the liquid sample by dielectrophoresis. An optical measurement method for generating different deformation diffracted light patterns and measuring information on particles in a liquid sample from the deformed diffracted light patterns, wherein the liquid contains at least two different kinds of particles. The sample is put in a container and applied to the electrode pair while continuously changing the applied voltage value so that the voltage value becomes gradually larger. In the liquid based on the change data of the deformation diffracted light pattern intensity at that time Measure information about contained particles.
これによれば、上述した光学的測定装置と同様の手順で、試料液体中の２種類あるいはそれ以上の数の異なる種類の粒子による変形回折光パターン強度（変形回折光パターンの回折光強度）を、それぞれ粒子ごとに分離して計測することができるので、混在した粒子の情報だけではなく、粒子ごとの情報を得ることができる。 According to this, the deformation diffracted light pattern intensity (diffracted light intensity of the deformed diffracted light pattern) by two or more different types of particles in the sample liquid is obtained in the same procedure as the optical measuring apparatus described above. Since each particle can be measured separately, not only information on the mixed particles but also information on each particle can be obtained.
以下、本発明の実施形態について図面を用いて説明する。なお、本発明は、以下に説明するような実施形態に限定されるものではなく、本発明の趣旨を逸脱しない範囲で種々の態様が含まれることはいうまでもない。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the embodiments described below, and it goes without saying that various aspects are included without departing from the spirit of the present invention.
図１は、本発明の一実施形態である光学的測定装置の構成を示す概略断面図、図２はその上面図である。
この実施形態の光学的測定装置は、誘電泳動を行いながら、光学的測定を行うものであり、液体試料を保持する容器１１、容器１１の底面となる底板１２aに形成され、回折格子を形成する一対の電極１３、１４と、電極１３および電極１４に交流電圧を印加する交流電源１５と、光源１６と、光源光を収束するレンズ光学系１７と、回折光を検出する光検出器１８と、印加電圧を制御する電圧制御部２０とからなる。
FIG. 1 is a schematic cross-sectional view showing a configuration of an optical measuring apparatus according to an embodiment of the present invention, and FIG. 2 is a top view thereof.
The optical measurement apparatus according to this embodiment performs optical measurement while performing dielectrophoresis, and is formed on a container 11 that holds a liquid sample and a bottom plate 12a that is a bottom surface of the container 11, and forms a diffraction grating. A pair of electrodes 13, 14, an AC power source 15 that applies an AC voltage to the electrodes 13 and 14, a light source 16, a lens optical system 17 that converges the light source light, and a photodetector 18 that detects diffracted light, The voltage control unit 20 controls the applied voltage.
容器１１は、底板１２ａの上に、側壁となる枠体１２ｂを貼り付けることにより形成してある。この容器１１は、ガラス等の光透過性の材料が用いられ、底板１２ａを通して、入射光が電極１３、１４（回折格子）に照射できるようにしてある。 The container 11 is formed by sticking a frame body 12b serving as a side wall on the bottom plate 12a. The container 11 is made of a light-transmitting material such as glass, so that incident light can be applied to the electrodes 13 and 14 (diffraction grating) through the bottom plate 12a.
電極１３、１４は、マスクパターニング手法を用いて，底板１２ａ上に形成される。なお、本実施形態では、底板１２ａに電極１３、１４を形成しているが、容器１１が十分に深い場合には、底板１２ａに代えて、側壁となる枠体１２ｂに電極１３、１４を形成してもよい。
液体試料をこの容器１１に入れ、電極１３、１４を液体中に浸すことにより、電極１３、１４と液体試料とが接するようになる。なお、電極表面に耐食性の保護膜（例えばＳｉＯ２）を形成した場合は、液体と電極とは非接触となるが、このような場合も実質的に接するものとして扱ってよい。要するに、電極に印加する電圧により誘電泳動を生じさせることができればよい。
電極１３は、２本の平行な直線状電極片１３ａ、１３ｂが隣接する直線状電極片偏在領域１３ｆと、直線状電極片形成されていない直線状電極片不在領域１３ｃとが、交互に繰り返すようにしてあり、すべての直線状電極片１３ａ、１３ｂは、接続部１３ｄにより電気的に接続され、いわゆる櫛型電極構造としてある。
電極１４についても同様であり、２本の平行な直線状電極片１４ａ、１４ｂが隣接する直線状電極片偏在領域１４ｆと、直線状電極片が形成されていない直線状電極片不在領域１４ｃとが、交互に繰り返すようにしてあり、すべての直線状電極片１４ａ、１４ｂは、接続部１４ｄにより電気的に接続され、櫛型電極構造としてある。
The electrodes 13 and 14 are formed on the bottom plate 12a using a mask patterning technique. In the present embodiment, the electrodes 13 and 14 are formed on the bottom plate 12a. However, when the container 11 is sufficiently deep, the electrodes 13 and 14 are formed on the frame 12b serving as the side wall instead of the bottom plate 12a. May be.
By putting the liquid sample into the container 11 and immersing the electrodes 13 and 14 in the liquid, the electrodes 13 and 14 come into contact with the liquid sample. In addition, when a corrosion-resistant protective film (for example, SiO 2 ) is formed on the electrode surface, the liquid and the electrode are not in contact with each other, but such a case may be handled as being substantially in contact. In short, it is sufficient that dielectrophoresis can be caused by the voltage applied to the electrodes.
In the electrode 13, linear electrode piece unevenly distributed regions 13f adjacent to two parallel linear electrode pieces 13a and 13b and linear electrode piece absent regions 13c in which no linear electrode pieces are formed are alternately repeated. All of the linear electrode pieces 13a and 13b are electrically connected by the connecting portion 13d, and have a so-called comb electrode structure.
The same applies to the electrode 14, and a linear electrode piece unevenly distributed region 14f in which two parallel linear electrode pieces 14a and 14b are adjacent to each other and a linear electrode piece absent region 14c in which no linear electrode pieces are formed are provided. The linear electrode pieces 14a and 14b are electrically connected to each other through the connecting portion 14d to form a comb electrode structure.
そして、電極１３の直線状電極片不在領域１３ｃの位置に、電極１４の直線状電極片１４ａ、１４ｂがくるように配置して、電極１３の直線状電極片１３ａ、１３ｂと、電極１４の直線状電極片１４ａ、１４ｂとが、等間隔で連続的に並ぶようにして、直線状電極片１３ａ、１３ｂ、１４ａ、１４ｂにより回折格子が形成されるようにしてある。 Then, the linear electrode pieces 14 a and 14 b of the electrode 14 are arranged at the position of the linear electrode piece absent region 13 c of the electrode 13, and the straight electrode pieces 13 a and 13 b of the electrode 13 and the straight line of the electrode 14 are arranged. The linear electrode pieces 13a, 13b, 14a, and 14b form a diffraction grating so that the electrode pieces 14a and 14b are continuously arranged at regular intervals.
回折格子の寸法は、回折格子を構成する電極幅ｄ１、電極間隔ｄ２のいずれについても、０．５μｍ〜２０μｍ程度にするのが好ましいが、回折光を発生させることができるものであれば、形状や寸法は、特に限定されない。例えば、電極幅ｄ１と電極間隔ｄ２とが異なる寸法になるようにしてもよいし、回折格子形状が直線状電極片で構成されなくてもよい。なお、本実施形態の回折格子では、電極幅１０μｍ、電極間隔１０μｍの直線状電極片としている。この場合、回折格子の格子間隔ｄは、ｄ１＋ｄ２となる。 The size of the diffraction grating is preferably about 0.5 μm to 20 μm for both the electrode width d1 and the electrode interval d2 constituting the diffraction grating. However, the shape of the diffraction grating is not limited as long as it can generate diffracted light. The dimensions are not particularly limited. For example, the electrode width d1 and the electrode interval d2 may have different dimensions, or the diffraction grating shape may not be formed of a linear electrode piece. In the diffraction grating of the present embodiment, a linear electrode piece having an electrode width of 10 μm and an electrode interval of 10 μm is used. In this case, the grating interval d of the diffraction grating is d1 + d2.
交流電源１５には、電圧０から（第１臨界点の予測がつく場合はそれより小さい値からでよい）、連続的に印加電圧値を増大するように変化することができる交流電源が用いられる。具体的には、０〜１００Ｖで可変であり、周波数が１０ＫＨｚ〜１０ＭＨｚ程度の交流電圧が印加できる交流電源を使用する。なお、一般的には、高周波電源を用いるのが好ましい。 The AC power source 15 is an AC power source that can change from a voltage of 0 (or a smaller value if the first critical point can be predicted) to continuously increase the applied voltage value. . Specifically, an AC power supply that is variable from 0 to 100 V and that can apply an AC voltage with a frequency of about 10 KHz to 10 MHz is used. In general, it is preferable to use a high-frequency power source.
光源１６は、測定対象となる液体試料に応じて種類を選択すればよいが、例えば、Ｈｅ−Ｎｅレーザ光源（波長６３３ｎｍ）や、その他のレーザ光源を用いるのが好ましい。
レンズ光学系１７は、光源光を収束して、電極１３、１４（回折格子）に照射できるように構成してある。なお、光源光の入射角度を調整できるようにして、測定対象、測定目的に応じて、透過回折光、反射回折光のいずれでも、取得できるようにするのが好ましい。
透過回折光を測定する場合、入射角は、容器底面と液体試料との界面で全反射が生じない条件であればよく、例えば、入射角０度で入射させてもよい。
The type of the light source 16 may be selected according to the liquid sample to be measured. For example, it is preferable to use a He—Ne laser light source (wavelength 633 nm) or another laser light source.
The lens optical system 17 is configured to converge the light source light and irradiate the electrodes 13 and 14 (diffraction grating). In addition, it is preferable that the incident angle of the light source light can be adjusted so that either transmitted diffracted light or reflected diffracted light can be obtained according to the measurement object and the measurement purpose.
When measuring transmitted diffracted light, the incident angle may be a condition that does not cause total reflection at the interface between the bottom surface of the container and the liquid sample. For example, the incident angle may be incident at an incident angle of 0 degree.
光検出器１８は、透過回折光を検出するため、液体試料の上部側に配置する。光検出器１８には、回折角を測定するための角度調整機構が設けられており、回折光の強度とともに回折角が検出できるようにしてある。この光検出器１８には、フォトダイオードやＣＣＤが用いられる。なお、角度調整機構を設ける代わりに、複数の素子を並べたアレイセンサを用いて、回折角が計測できるようにしてもよい。 The photodetector 18 is arranged on the upper side of the liquid sample in order to detect the transmitted diffracted light. The light detector 18 is provided with an angle adjusting mechanism for measuring the diffraction angle so that the diffraction angle can be detected together with the intensity of the diffracted light. A photodiode or CCD is used for the photodetector 18. Instead of providing the angle adjustment mechanism, the diffraction angle may be measured using an array sensor in which a plurality of elements are arranged.
電圧制御部２０は、交流電源１５の出力電圧値を制御して、電極対１３、１４への印加電圧を制御する。 The voltage control unit 20 controls the output voltage value of the AC power supply 15 to control the voltage applied to the electrode pairs 13 and 14.
次に、上記装置の計測動作について説明する。ここでは、液体試料中に、２種類の異なる粒子Ｓ１、Ｓ２が混在しているものとする。説明を簡略化するため、Ｓ１、Ｓ２は同一材料で粒径が異なる（粒径はＳ１＞Ｓ２）ものとする。
まず、電極１３、１４に、電圧を印加しない状態で、光源１６から入射光を照射する。液体試料は、粒子Ｓ１、Ｓ２が拡散し、全体がほぼ均一な状態になっている。このとき、入射光は、直線状電極片１３ａ、１３ｂ、１４ａ、１４ｂにより形成される回折格子（以下、周期電極による回折格子（周期ｄ）という）の影響を受け、図３において実線で示すように、周期ｄの回折条件を満たす角度位置に、−１次、０次、１次、・・・の透過回折光による回折光パターン（基本回折光パターンという）が発生する。
Next, the measurement operation of the above apparatus will be described. Here, it is assumed that two different types of particles S1 and S2 are mixed in the liquid sample. In order to simplify the description, it is assumed that S1 and S2 are the same material and have different particle sizes (particle size is S1> S2).
First, incident light is irradiated from the light source 16 to the electrodes 13 and 14 without applying a voltage. In the liquid sample, the particles S1 and S2 are diffused, and the whole is in a substantially uniform state. At this time, incident light is affected by a diffraction grating formed by the linear electrode pieces 13a, 13b, 14a, and 14b (hereinafter referred to as a diffraction grating (period d) by a periodic electrode), and as shown by a solid line in FIG. In addition, a diffracted light pattern (referred to as a basic diffracted light pattern) by transmitted diffracted light of −1st order, 0th order, 1st order,.
次に、交流電源１５により、電極１３、電極１４間に交流電圧を印加する。液体試料中に粒子Ｓ１、Ｓ２が存在すると、交流電圧が十分に高い場合には粒子の拡散力より誘電泳動力が勝ることにより、誘電泳動作用が働き、粒子Ｓ１、Ｓ２は電気力線が集中する領域に移動する。図４は、交流電圧を印加したときの粒子の状態を説明する図である。図に示すように、正極と負極とが隣接することにより電気力線が集中する、直線状電極片１４ｂと直線状電極片１３ａとの間、あるいは、直線状電極片１３ｂと直線状電極片１４ａとの間に、粒子が集中するようになり、屈折率が高い粒子集中領域Ｐが形成される。この屈折率が高い粒子集中領域Ｐは、格子間隔ｄの２倍の周期（２ｄ）で発生しており、このときの屈折率分布による周期２ｄの回折格子を形成する。
入射光は、周期電極による回折格子（周期ｄ）の影響を受けて、基本回折光パターンを発生するとともに、屈折率分布による回折格子（周期２ｄ）の影響を受けて、図３に破線で示すように、周期２ｄの回折条件を満たす角度位置に、−１次、１次、・・・の透過回折光による回折光パターンが追加された変形回折光パターンを発生する。（ただし、０次透過回折光は、基本回折光パターンにおける０次の回折光と重なる）。
Next, an AC voltage is applied between the electrode 13 and the electrode 14 by the AC power source 15. When particles S1 and S2 are present in the liquid sample, if the alternating voltage is sufficiently high, the dielectrophoretic force is greater than the diffusive force of the particles, thereby causing a dielectrophoretic action, and the lines of electric force are concentrated on the particles S1 and S2. Move to the area you want. FIG. 4 is a diagram for explaining the state of particles when an AC voltage is applied. As shown in the figure, the lines of electric force concentrate due to the adjacent positive and negative electrodes, or between the linear electrode piece 14b and the linear electrode piece 13a, or between the linear electrode piece 13b and the linear electrode piece 14a. In between, the particles are concentrated, and the particle concentration region P having a high refractive index is formed. The particle concentration region P having a high refractive index is generated with a period (2d) twice as long as the grating interval d, and forms a diffraction grating with a period 2d by the refractive index distribution at this time.
The incident light is influenced by the diffraction grating (period d) by the periodic electrode to generate a basic diffracted light pattern and is also influenced by the diffraction grating (period 2d) by the refractive index distribution, and is shown by a broken line in FIG. As described above, a deformed diffracted light pattern is generated by adding a diffracted light pattern of -1st order, 1st order,... Transmitted diffracted light at an angular position satisfying the diffraction condition of the period 2d. (However, the 0th-order transmitted diffracted light overlaps the 0th-order diffracted light in the basic diffracted light pattern).
粒子集中による屈折率分布により生じた回折光パターン（図３の破線の±１次光）の回折光強度は、捕集された粒子Ｓ１、Ｓ２の密度に依存する。したがって、交流印加電圧値を０または十分に小さい値から次第に大きくなるように変化させ、粒子集中領域の粒子密度を変化させ、このときの回折光強度を検出するようにする。 The diffracted light intensity of the diffracted light pattern (± first-order light of the broken line in FIG. 3) generated by the refractive index distribution due to particle concentration depends on the density of the collected particles S1 and S2. Therefore, the AC applied voltage value is changed so as to gradually increase from 0 or a sufficiently small value, the particle density in the particle concentration region is changed, and the diffracted light intensity at this time is detected.
図５および図６は、印加電圧値を変化させながら回折光強度（図３の破線の±１次光）を測定し、印加電圧値の二乗に対してこの回折光強度の変化を示したデータである。なお、図５は、参考とするため粒子Ｓ１と粒子Ｓ２とを別々に測定したときのデータであり、図６は、２種類の粒子Ｓ１、Ｓ２が混在する試料液体について測定したデータである。 FIG. 5 and FIG. 6 show data indicating the change in the diffracted light intensity with respect to the square of the applied voltage value by measuring the diffracted light intensity (± first-order light of the broken line in FIG. 3) while changing the applied voltage value. It is. Note that FIG. 5 is data when the particles S1 and S2 are separately measured for reference, and FIG. 6 is data measured for a sample liquid in which two types of particles S1 and S2 are mixed.
図５に示すように、粒子Ｓ１を含む液体試料では、印加電圧値がＡ点（第１臨界点）に達すると、誘電泳動による粒子集中が始まり、回折光強度が増加する。そしてＣ点（飽和点）は、捕集できる粒子Ｓ１がすべて捕集され、回折光強度が最大となった状態である。同様に、粒子Ｓ２を含む液体試料では、印加電圧値がＢ点（第２臨界点）に達すると、誘電泳動による粒子集中が始まり、回折光強度が増加する。そしてＤ点（飽和点）は、捕集できる粒子Ｓ２がすべて捕集され、回折光強度が最大となった状態である。 As shown in FIG. 5, in the liquid sample containing the particles S1, when the applied voltage value reaches the point A (first critical point), particle concentration by dielectrophoresis starts and the diffracted light intensity increases. Point C (saturation point) is a state in which all the particles S1 that can be collected are collected and the diffracted light intensity is maximized. Similarly, in the liquid sample containing the particles S2, when the applied voltage value reaches the point B (second critical point), particle concentration by dielectrophoresis starts and the diffracted light intensity increases. Point D (saturation point) is a state where all the particles S2 that can be collected are collected and the intensity of diffracted light is maximized.
これら２種類の液体試料を適当な比で混合して、同様の測定を行うと図６に示すように回折光強度は図４のＡ、Ｂ、Ｃ、Ｄ点と同じ電圧値で変曲するようになる。このことから図５においてもＡ、Ｂ点は粒子Ｓ１、Ｓ２それぞれの臨界点であり、Ｃ、Ｄ点は粒子Ｓ１、Ｓ２それぞれの飽和点であることがわかる。 When these two types of liquid samples are mixed at an appropriate ratio and the same measurement is performed, the intensity of diffracted light is inflected at the same voltage value as points A, B, C, and D in FIG. 4 as shown in FIG. It becomes like this. From this, also in FIG. 5, points A and B are the critical points of the particles S1 and S2, respectively, and points C and D are the saturation points of the particles S1 and S2.
図６の粒子Ｓ１の回折光強度が飽和したときの回折光強度値ＩＳ１と粒子Ｓ２の回折光強度が飽和したときの回折光強度値ＩＳ２との和は、液体試料中に存在する粒子Ｓ１、粒子Ｓ２のすべてを捕集したときの回折光強度である。
粒子Ｓ１と粒子Ｓ２とが同じ材料であって、屈折率が同じであれば、その混合比率は粒子Ｓ１の飽和回折光強度値ＩＳ１と粒子Ｓ２による飽和回折光強度値ＩＳ２との比に比例することになる。
なお、粒子Ｓ１と粒子Ｓ２との材質が異なるときは、予めそれぞれの粒子Ｓ１、Ｓ２を単独で含む標準試料を用いて回折光強度に寄与する比である係数を求めておけばよい。
このようにして、印加電圧値を変化させながら回折光強度を測定したときの変曲点（臨界点、飽和点）を求めることにより、混在する粒子を分離して、それぞれの回折光強度を求めることができる。
The sum of the diffracted light intensity value I S2 of when diffracted light intensity is saturated diffracted light intensity values I S1 and particles S2 of when diffracted light intensity is saturated particles S1 in FIG. 6, the particles present in a liquid sample This is the diffracted light intensity when all of S1 and particle S2 are collected.
If the particle S1 and the particle S2 are the same material and have the same refractive index, the mixing ratio is the ratio of the saturation diffracted light intensity value I S1 of the particle S1 to the saturated diffracted light intensity value I S2 of the particle S2. It will be proportional.
In addition, when the material of particle | grains S1 and particle | grains S2 is different, what is necessary is just to obtain | require the coefficient which is a ratio which contributes to diffracted light intensity | strength previously using the standard sample which contains each particle | grains S1 and S2 independently.
In this way, by obtaining the inflection point (critical point, saturation point) when the diffracted light intensity is measured while changing the applied voltage value, the mixed particles are separated and the respective diffracted light intensity is obtained. be able to.
さらに、予め、標準試料により、回折光強度と濃度、粒径、粒形、結合状態との関係を測定して基礎データを持つことにより、基礎データの比較により、それぞれの粒子についての濃度や結合状態の情報を得ることもできる。 Furthermore, by measuring the relationship between diffracted light intensity and concentration, particle size, particle shape, and binding state with a standard sample in advance and having basic data, the concentration and binding of each particle can be compared by comparing the basic data. You can also get status information.
また、上記実施形態では液体中に含まれる粒子は２種類としたが、３種類以上であっても、計算がやや複雑にはなるが、同様の方法で回折光強度を粒子ごとに分離することができる。 In the above embodiment, the number of particles contained in the liquid is two. However, even if there are three or more types, the calculation is slightly complicated, but the diffracted light intensity is separated for each particle by the same method. Can do.
また、上記実施形態では、電極対は、正の電極と負の電極とが隣接する部分が、回折格子周期の２倍以上の整数倍の周期で繰り返すようにするために、一定間隔で並ぶ直線状の電極片１３ａ、１３ｂと、直線状電極片１４ａ、１４ｂとが交互に配置されるようにしたが、電極パターンはこれに限られず、他のパターンであってもよい。 In the above embodiment, the electrode pair is a straight line arranged at regular intervals so that the portion where the positive electrode and the negative electrode are adjacent is repeated at a period that is an integral multiple of twice or more the diffraction grating period. The electrode pieces 13a and 13b and the linear electrode pieces 14a and 14b are alternately arranged. However, the electrode pattern is not limited to this, and other patterns may be used.
本発明は、２種類以上の粒子が混在する液体試料について測定する液体試料中の粒子の光学的測定を行う光学的測定装置などに利用することができる。 The present invention can be used for an optical measurement device that performs optical measurement of particles in a liquid sample for measuring a liquid sample in which two or more kinds of particles are mixed.
１１： 容器
１３、１４ 電極
１５： 交流電源
１６： 光源
１８： 光検出器
２０：電圧制御部
11: Containers 13 and 14 Electrode 15: AC power supply 16: Light source 18: Photo detector 20: Voltage control unit
Claims (3)
電極対に電圧を印加して液体試料の屈折率分布を変化することにより、基本回折光パターンとは異なる変形回折光パターンを発生させるとともに、電圧制御部により印加電圧値が連続的に大きくなるように変化させたときの変形回折光パターン強度の変化に基づいて液体試料に関する情報を計測することを特徴とする光学的測定装置。 A basic diffracted light pattern is formed by irradiating light from a light source, an AC power source capable of changing the applied voltage value, a container holding a liquid sample, and a liquid sample in the container. The generated diffraction grating, an electrode pair that constitutes at least a part of the diffraction grating and can be applied with an AC voltage from an AC power source, a voltage control unit that changes a voltage value applied to the electrode pair, and a diffraction grating A photodetector for detecting diffracted light,
By applying a voltage to the electrode pair to change the refractive index distribution of the liquid sample, a modified diffracted light pattern different from the basic diffracted light pattern is generated, and the applied voltage value is continuously increased by the voltage controller. An optical measuring device that measures information about a liquid sample based on a change in the intensity of the deformed diffracted light pattern when the pattern is changed to.
少なくとも２種類の異なる粒子が混在する液体試料を容器内に入れ、
印加電圧値を次第に大きい電圧値になるように連続的に変化させながら電極対に印加して、そのときの変形回折光パターン強度の変化のデータを光検出器により計測し、
変形回折光パターン強度の変化のデータに基づいて液体中に含まれる粒子に関する情報を計測することを特徴とする光学的測定方法。 A basic diffracted light pattern is formed by irradiating light from a light source, an AC power source capable of changing the applied voltage value, a container holding a liquid sample, and a liquid sample in the container. The electrode pair includes a generated diffraction grating, an electrode pair that constitutes at least part of the diffraction grating and can be applied with an AC voltage from an AC power source, and a photodetector that detects diffracted light from the diffraction grating. An optical measurement method for measuring information about particles in a liquid sample from the deformed diffracted light pattern by generating a deformed diffracted light pattern different from the basic diffracted light pattern by applying an alternating voltage to change the refractive index distribution of the liquid sample. Because
Put a liquid sample containing at least two different types of particles in the container,
Applying to the electrode pair while continuously changing the applied voltage value so as to gradually increase the voltage value, and measuring the data of the deformation diffracted light pattern intensity at that time by a photodetector,
An optical measurement method, comprising: measuring information related to particles contained in a liquid based on data of a change in deformation diffracted light pattern intensity.
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